European Journal of. Immunology. Supporting Information for. DOI 10.1002/eji.201343429. Christina Hesse, Wiebke Ginter, Theresa F¨org, Christian T. Mayer,.
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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201343429 Christina Hesse, Wiebke Ginter, Theresa Forg, Christian T. Mayer, ¨ Abdul Mannan Baru, Catharina Arnold-Schrauf, Wendy W. J. Unger, Hakan Kalay, Yvette van Kooyk, Luciana Berod and Tim Sparwasser In vivo targeting of human DC-SIGN drastically enhances CD8+ T-cell-mediated protective immunity
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
Supplemental Information In vivo targeting of human DC-SIGN drastically enhances CD8+ T cellmediated protective immunity Christina Hesse, Wiebke Ginter, Theresa Förg, Christian T. Mayer, Abdul Mannan Baru, Catharina Arnold-Schrauf1, Wendy W. J. Unger, Hakan Kalay, Yvette van Kooyk, Luciana Berod and Tim Sparwasser
OVA -DTT
1500
1000
Conjugate
250 150 100 75
500
50
VA O
25
SSC-Lin
15 +AZN-D1
Lymphocytes 60K
40K
40K
59.45 20K
66.63
20K
0
0 0
20K
40K
60K
0
FSC-Lin +AZN-D1
0.5
5
15
soluble OVA (µg/mL)
AZN-D1:OVA
+AZN-D1
10
4
10
3
10
2
20K
40K
10
1
10
0
60K
10
4
10
3
10
2
VA A
-D ZN
O 1:
VA
Live Cells
95.34
10
0
SSC Area
10
1
10
2
3
10
10
4
CFSE 400 300
DAPI
5 0
Single Cells
60K
DAPI
WT hSIGN
Medium
# CFSElow OT-II+ / Well (x103)
D
10
1
10
0
93.86
# Cells
C
:O pe y ot Is
SSC-Lin
OVA
10
0
OVA
37
20
Raji WT Raji DC-SIGN
MFI
Conjugate IgG1
B
AZN-D1 +DTT
AZN-D1
Isotype
AZN-D1:OVA
Isotype:OVA
OVA 1 µg
10 µg
AZN-D1:OVA -DTT
A
AZN-D1:OVA +DTT
Suppl.Fig1
200
69.56
30.44
100
100
101
CD4
102
103
104
0 100
101
102
103
104
CFSE
Suppl.Fig.1 (A) Chemical conjugation of OVA to the AZN-D1 and isotype control antibodies was verified using SDS-gel electrophoresis and silverstaining (left panel) or Western blot (right panel) using anti-OVA (mouse, Ig2a) primary antibody and rabbit-anti-mouse Ig2a secondary antibody (B) Binding of αDCSIGN:OVA (AZN-D1:OVA), isotype:OVA and soluble OVA protein to human DC-SIGN (black bars) or mock transfected (grey bars) Raji cells. Transfected Raji cells were incubate with 10µg/mL of AZN-D1:OVA, isotype:OVA or soluble OVA. Cells were washed and stained with a fluorescent labelled secondary goat-αmouse IgG1 antibody. The mean fluorescence intensity is compared (C) WT or hSIGN BMDCs were loaded with different concentrations of soluble OVA protein with or without 0.5µg/mL unconjugated αDC-SIGN antibody (AZN-D1), medium or 0.5µg/mL αDC-SIGN antibody conjugated to OVA (AZN-D1:OVA). Proliferation of CD4+OT-II cells was assessed using CFSE dilution. The number of CFSElowCD4+OT-II T cells is displayed. (D) Progressive gating strategy for the identification of proliferating CD4+OT-II and CD8+OT-I T cells (Suppl.Fig.1C)
Suppl.Fig 2 Supplementary to figure 1 A Lymphocytes Single Cells 30K
Live Cells
Proliferating cells
30K
30K
SSC-Lin
10K
0
10K
20K
0
10K
20K
30K
96.18 0
SSC Area
10
1
10 OT-II 8 6 4 2
10
3
0 10
1
10
2
10
3
0 0
10
CD4 / CD8
10
1
10
2
10
3
10
4
CFSE
14 OT-I 12 10 8 6 4 2 0
M
Concentration in µg/mL
2
101
0
1
0. 5
0. 1
1
5 0. 0
0. 0
0. 0
05
0
M ed i
2
10
eFl450
C
um
# CFSElow OT-II+ / Well (x104)
FSC-Lin
B
0
0
30K
10
10K
# CFSElow OT-I+ / Well (x104)
92.18
0
2
101
Aqua
10K
10
84.54
103 20K
SSC-Area
20K
SSC-Lin
20K
CD4 / CD8
103
m 05 .01 .05 iu .0 0 0 d 0 e
1 0.
5 0.
1
Concentration in µg/mL
WT + isotype:OVA + CD40 WT + DC-SIGN:OVA + CD40 hSIGN + isotype:OVA + CD40 hSIGN + DC-SIGN:OVA + CD40
Suppl.Fig.2 (A) Progressive gating strategy for the identification of proliferating CD4+OT-II T cells (Fig.1C and Suppl.Fig.2B,C). (B,C) Total numbers of (B) CFSElowCD4+OT-II and (C) CFSElowCD8+OT-I T cells cocultured with pulsed WT or hSIGN DCs receiving increasing doses of αDC-SIGN:OVA or isotype:OVA antibody plus αCD40. Mean of duplicate wells from one out of three experiments is depicted.
Suppl.Fig.3 A
C Counts
WT hSIGN 92.8%
200K 150K
SSC-A
8.4%
250K
100K
cDC
pDC
50K
74.70
0 0
hDC - SIGN
B
50K 100K 150K 200K 250K
FSC-A 250K
10
5
10
4
10
3
200K
CD8
80.75
100K
Aqua
96.0%
SSC-A
150K
10
50K
2
0 0 0
99.9%
SiglecH
85.9%
10
5
10
4
10
3
10
2
103
104
105
Linage neg
pDC cDC CD8
Counts
SSC-W
CD4
18.08 0 102
50K 100K 150K 200K 250K
0
10
5
10
4
10
3
10
2
0 0 10
2
CD11c
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
CD4
hDC - SIGN
Suppl.Fig.3 (A) Expression of human DC-SIGN on cDCs (CD11chighSiglecH-, left) and pDCs (CD11cintSiglecH+, right). Human DC-SIGN is expressed on hSIGN (black line), but not on WT (grey) DCs. (B) Expression of human DC-SIGN on cDC subsets (CD4+, CD8+ and double negative CD11c+ cDCs), One representative of three experiments. (C) Progressive gating strategy for the identification of different DC subsets. Linage positive cells are defined by NK1.1, CD19 and CD3 expression (Suppl.Fig.3A,B).
Suppl. 4 Supplementary to Fig.2 Lymphocytes
20K
20K
30K
10K
23.13
0 0
FSC-Lin
20K
30K
84.33 10K
SSC Area
2 1 3 5 7 9 Days post-immunisation
20K
10
1
30K
C
3
1
2
99.03
0 0
4 OT-II
0
10
0
% OT-I+ of total CD8+
% OT-II+ of total CD4+
B
10K
10K
3
PI
SSC-Lin
SSC-Lin
10
11
0
10
PE
1
10
2
10
3
CD45.1
30K
Transferred Cells
Live Cells
Single Cells
30K
SSC-Area
A
20K
10K
10
3
10
2
10
1
0 0 0 10
10
1
10
2
CD4 / CD8
10
3
10
4
10
0
10
1
10
2
CD4 / CD8
10
3
10
4
30 OT-I 20 hSIGN+ Isotype:OVA
10
hSIGN+ Isotype:OVA + CD40 hSIGN+ DC-SIGN:OVA
0
hSIGN+ DC-SIGN:OVA + CD40
1
3 5 7 9 11 Days post-immunisation
Suppl.Fig.4 (A) Progressive gating strategy for the identification of adoptively transferred CD45.1+CD4+OT‑II or CD45.1+CD8+OT‑I T cells (Fig.2 and Suppl.Fig.4B,C). (B,C) Expansion of adoptively transferred (B) CD45.1+CD4+OT‑II or (C) CD45.1+CD8+OT‑I in the blood of WT or hSIGN mice treated with αDC-SIGN:OVA or isotype:OVA with or without αCD40 on day 1 to 11 post-immunisation. 3x105 enriched CD4+OT-II or CD8+OT-I T cells were transferred. Mean of 2 mice/group. One of two experiments performed.
Supp.Fig. 5 Supplementary to Fig. 3 + CD40
***
0.1%
0.3%
hSIGN 0.04%
0.04%
100 n.s.
80
MOG35-55
60 40
72.0%
50.1%
72.2%
99.2%
20
DC-SIGN:OVA CD40
C
B
WT
+ -
+ -
+ +
WT
hSIGN
Lymphocytes
SIINFEKL
+ +
IFN
% IFN
+
of CD45.1+CD8+
A
hSIGN
WT
CD8
Single Cells
Live Cells
FSC-Lin
# Cells
PI
SSC-Area
80.51
SSC-Lin
SSC-Lin
72.90
81.52
SSC Area
CFSE
46.88
53.12
6.65
CFSE
CFSE
Suppl.Fig.5 (A) Frequency of IFNγ-producing CD45.1+CD8+OT‑I T cells in immunised WT and hSIGN mice after ex vivo re-stimulation with SIINFEKL peptide. Bar graphs represent frequencies from pooled data from two experiments. (B) Representative dot plots from data shown in Fig.3 and Suppl.Fig.5A re-stimulated with SIINFEKL as well as re-stimulation with an irrelevant peptide (MOG35-55). (C) Progressive gating strategy used to calculate specific killing of SIINFEKL-loaded spleen cells (Fig.3B,C).
SUPP 6 A
Lymphocytes
Single Cells
Live Cells
EMA
SSC-A
SSC-A
92.90
52.64
98.97
FSC-A
SSC-H
PE
8.13
CD8
SIINFEKL
11.60
SIINFEKL
CD62L
Suppl.Fig.6 (A) Progressive gating strategy for the identification of CD62Llowpentamer+CD8+ T cells of immunised WT and hSIGN after infection (Fig.4B,C and Fig.5E).
Supplementary to Figure 5
1.0
IgG2c
OD405 nm
0.8 0.6 0.4 0.2 0.0
*
B
10
CFU / Liver (log10)
A
1:500
1:2000 1:4000 1:8000 Dilution
WT non-vaccinated WT vaccinated hSIGN non-vaccinated hSIGN vaccinated
8 6 4 n.s.
2
DC-SIGN:OVA CD40 OVA:IFA boost
n.s.
WT
+
+ + +
+ + +
+ + -
hSIGN
Suppl.Fig.7 (A) OVA-specific IgG2c titration in the serum of vaccinated WT and hSIGN mice measured by ELISA. (B) Bacterial burden in the spleen of vaccinated WT and hSIGN mice 4 days after infection with LMOVA (2x105 CFU) and 39 days after initial immunisation.
Supplementary Methods Binding Assay Stable Raji transfectants expressing the human DC-SIGN receptor (Raji DC-SIGN) were generated as previously described in (Geijtenbeek et al., 2000). Raji or Raji- DC-SIGN cells were then incubated with 10µg/ml soluble OVA, AZN-D1:OVA or isotype:OVA at 4°C for 30min. After washing, cells were incubated with APC-labelled goat-anti-mouse IgG1 antibody (GaM-IgG-AF647, Molecular Probes) for 30min on ice. The mean fluorescence intensity was determined by flow cytometry (Facs Calibur, BD Biosciences). Western Blot and Silverstain Samples were boiled in Laemmli sample buffer before proteins were separated on a 7.5-8% sodium dodecyl sulfate (SDS)-gel and transferred by electroblotting onto PVDF membranes (Immobilon-P, Millipore). Membranes were blocked with 5% dry milk and incubated with primary antibodies detecting OVA (IgG2a, clone: 6G2, AntibodyShop). Membranes were washed in 0.1% Tween/Tris(hydroxymethyl)aminomethane– buffered saline and incubated with the respective horseradish peroxidase–conjugated secondary antibody: rabbit-anti–mouse-immunoglobulin (anti-IgG2a, Invitrogen). Detection was performed using enhanced chemiluminescence reagent (Pierce). Silverstaining was performed using the BioRad silverstain kit according to manufacturer’s instructions.
Publication Geijtenbeek, T.B., Kwon, D.S., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C., Middel, J., Cornelissen, I.L., Nottet, H.S., KewalRamani, V.N., Littman, D.R., et al. (2000). DC-SIGN, a dendritic cell-specific HIV-1binding protein that enhances trans-infection of T cells. Cell 100, 587-597.